WangLab:Cell Culture Protocol

Cell Culture Protocols:

Making Solutions:

• 10% FBS supplemented DMEM (1 Litre): warm up and add the following to 1 litre DMEM

              100% FBS 	       110 ml (10%)
              penecilin/streptomyosin 11 ml (1%)
              sodium pyruvate         11 ml (1%)
              L-glutamine             11 ml (1%)

• 0.5X trypsin in PBS (40ml): warm up and add 2 ml 10X trypsin-EDTA to 38 ml PBS
• 2% Gelatin (200 ml): get 200 ml MQ H2O, weigh 4 g Gelatin (cell culture grade) and add to H2O, gently shake bottle, put on a tray containing some water, loose the cap of the bottle, autoclave with liquid cycle (30min). After take out, be sure to shake the bottle until gelatin desolve uniformly in liquid, keep in refrigerator for later use.

Splitting Cells (1:3)

1. Rinse confluent cells with PBS for 3 times
2. Incubate cells with 2 ml 0.5x trypsin (10mm dish) (1x for smooth muscle cells and 0.5x for endothelial cells), keep in 37oC for 1.5 min, not to over 2 min.
3. gently pat the dish, check under microscopy to make sure all the cells are in suspension.
4. quickly add DMEM (must contain Calsium), collect cell solution into a tube and centrifuge 1000rpm for 3 min. (keep the balance of centrifuger).
5. During the centrifuging period, take 3 new tissue cultured dishes (if not tissue culture treated, coat the dishes with 2 % gelatin (just rinse), if not sterile, incubate with ethanol or light-bath with UV lamp for 30 min and then rinse with PBS for 3 times). Label the dishes with cell name, passage, date, initials of your name.
6. take out the centrifuged tube containing cells, you should be able to see a whitish pellet at the bottom of the tube. Tilt the tube and aspirate the supernatant with vacuum tip, resuspend the cell pellet with 10% FBS DMEM by pipetting up and down 20 times to break cell-cell aggregation. Apply cell solution to labeled dishes.
7. keep the cell dishes in the incubator supplemented with 5% CO2 at 37oC.

Freezing Cells

1. Thaw FBS, DMSO, Prepare freezing medium, 70% DMEM, 20% FBS, 10% DMSO (7ml, 2ml, 1ml for 10 ml medium), keep in 4oC.
2. Rinse confluent cells with PBS for 3 times
3. Incubate cells with 2 ml 0.5x trypsin (10mm dish) (1x for smooth muscle cells and 0.5x for endothelial cells), keep in 37oC for 1.5 min, not to over 2 min.
4. gently pat the dish, check under microscopy to make sure all the cells are in suspension.
5. quickly add DMEM (must contain Calsium), collect cell solution into a tube and centrifuge 1000rpm for 3 min. (keep the balance of centrifuger).
6. During the centrifuging period, Label the freeze epitaphs with cell name, passage, date, initials of your name.
7. take out the centrifuged tube containing cells, you should be able to see a whitish pellet at the bottom of the tube. Tilt the tube and aspirate the supernatant with vacuum tip, re-suspend the cell pellet with Freezing medium by pipetting up and down 20 times to break cell-cell aggregation. Apply cell solution to labeled epitaphs.
8. freeze in -70oC for overnight.
9. transfer the tubes to liquid nitrogen tank the next day, register in the log book.

Thawing Cells

1. Take out 1 epitaph from liquid nitrogen tank, thaw it in 37oC water bath until only little icy cube left. Take out the cell solution and Apply to 10% FBS DMEM.
2. collect cell solution into a tube and centrifuge 1000rpm for 3 min. (keep the balance of centrifuger).
3. During the centrifuging period, take new tissue cultured dishes (if not tissue culture treated, coat the dishes with 2 % gelatin (just rinse), if not sterile, incubate with ethanol or light-bath with UV lamp for 30 min and then rinse with PBS for 3 times). Label the dishes with cell name, passage, date, initials of your name.
4. take out the centrifuged tube containing cells, you should be able to see a whitish pellet at the bottom of the tube. Tilt the tube and aspirate the supernatant with vacuum tip, resuspend the cell pellet with 10% FBS DMEM by pipetting up and down 20 times to break cell-cell aggregation. Apply cell solution to labeled dishes.
5. keep the cell dishes in the incubator supplemented with 5% CO2 at 37oC.

Transfection

Lipofectamine 2000 method (Invitrogen) (for 1 well in 6-well cluster 10cm2)

1. the day before transfection, pass confluent cell 1:3 in 10%FBS without Penicillin/Straptomyosin.
2. check the cell condition, if it is in 60-80% confluency, proceed, otherwise, start a new experiment.
3. 1 mg DNA gently mixed into 100 ml Optimum only
4. 2 ul lipofectamine mixed into 100 ml Optimum only
5. Wait for 5 min, gently apply lipofectamine-Optimum to DNA-Optimum
6. mix thorough and gentle
7. incubate in RT for 20 min for the complex formation between DNA and lipofectamine
8. Apply the DNA-Lipofectamine complex Optimum to cells. Incubate 5.5 hr in incubator. check cell toxicity, if severe, stop the incubation and apply fresh 10%FBS-DMEM, if not, continue incubation until next day and change to 10%FBS DMEM.

Effectene method (Qiagen) (for 1 well in 6-well cluster 10cm2)

1. the day before transfection, pass confluent cell 1:3
2. check the cell condition, if it is in 40-80% confluency, proceed, otherwise, start a new experiment.
3. calculate before proceed (see Table 1)
4. add DNA in buffer EC, add enhancer, vortex 1-2 s, incubate 2-5 min, centrifuge
5. add effectene, vortex 10s, incubate 5-10 min at RT
6. aspirate cell medium, apply fresh medium
7. add fresh medium to DNA-effectene complex, pipette 2-3 times, apply onto cells, swirl a little to make sure DNAs are evenly distributed.
8. incubate for 6-18 hr. check cell toxicity, aspirate and apply fresh medium if necessary.

Table 1

names	DNA	Enhancer(8X DNA)	Buffer EC	Effectene(10X DNA)	10% FBS Medium to Cell	10% FBA Medium to complex
	1mg	8ml	100ml	10ml	1.6 ml	600 ml